M.1.

2_006

TREAT-NMD Activity A07: Accelerate preclinical phase of new therapeutic treatment

developement

Work package 7.4: Develop standardised protocols and procedures for harmonising and
accelerating pre-clinical studies (including standardised data analysis)

SOP Title Determination of hydroxyproline content as a

measure of fibrosis in nondystrophic and dystrophic
skeletal muscle.

SOP (ID) Number M.1.2_006

Version 1.0
Status Final/ November 25th 2009
Last reviewed November 25th 2009
Author C. George Carlson
Dept. Physiology, Kirksville College of Osteopathic
Medicine, Kirksville, MO. 63501-1497, USA
Working group Markus A. Rüegg (Biozentrum, University of Basel,
members Basel, Switzerland)
SOP responsible C. George Carlson
Official reviewer Markus A. Rüegg

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TABLE OF CONTENTS

OBJECTIVE.............................................................................................................................. 3
SCOPE AND APPLICABILITY .............................................................................................. 3
CAUTIONS............................................................................................................................... 4
MATERIALS ............................................................................................................................ 5
METHODS................................................................................................................................ 6
EVALUATION AND INTERPRETATION OF RESULTS.................................................... 9
REFERENCES........................................................................................................................ 11

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1 OBJECTIVE

The objective is to determine the total hydroxyproline expressed per mg of muscle

tissue wet weight (hydroxyproline content) as a quantitative measure of collagen deposition
and fibrosis. The procedure is adapted from Prockop and Udenfriend (1960) and Switzer and
Summer (1971). Briefly, individual muscles are weighed before being acid-hydrolyzed at
130°C for 12 hours in 5N HCl (10 mg muscle wet weight/ml). Samples of the hydrolysate
equivalent to 0.5 mg of muscle (50 µl) are diluted with 2.25 ml of distilled water and
neutralized with appropriate amounts of 0.1N KOH (phenolphthalein indicator). Sodium
borate buffer (0.5 ml, pH 8.7) is then added and the mixture is oxidized with 2.0 ml of 0.2 M
chloramine-T solution. After a brief incubation (25 minutes), the oxidation reaction is stopped
by adding 1.2 ml of 3.6 M sodium thiosulfate. Since the pyrroline and pyrrole carboxylic
oxidation products that are formed from hydroxyproline are not soluble in toluene,
contaminating impurities are extracted by adding 2.5 ml toluene and saturating amounts of
KCl (1.5 g). After appropriate mixing of the toluene and aqueous phases, the phases are
separated by centrifugation (300 to 400g for 1 minute). The toluene phase containing the
extracted impurities is then removed and discarded. The remaining aqueous layer containing
the hydroxyproline products is heated for 30 minutes in boiling water to convert the oxidation
product of hydroxyproline, pyrrole-2-carboxylic acid, to pyrrole. The final pyrrole reaction
product is then removed in a second toluene extraction, and 1.5 ml of the final toluene layer is
mixed with 0.6 ml Ehrlich’s reagent for colorimetric assay against hydroxyproline standards
(0.0, 0.5, 1.0, 2.0, 4.0 6.0 µg hydroxyproline) at 560 nm. Hydroxyproline contents of
individual muscles are expressed as µg hydroxyproline/mg muscle wet weight.

2 SCOPE AND APPLICABILITY

The method provides a quantitative scalar determination of the total amount of

hydroxyproline per mg muscle wet weight. The technique was first used to assess the
hydroxyproline levels in the mdx diaphragm (Stedman et al., 1991) and provides a simple and
relatively rapid quantitative determination that can be used to assess efficacy in reducing
fibrosis in dystrophic muscle.
Under some circumstances, this protocol for determining hydroxyproline content
should be complemented by independent histological determinations of fibrosis using a
trichrome staining technique (or suitable alternative) and point counting measures to assess
the proportion and distribution of fibrotic tissue within muscle cross sections. For example, in
some cases a particular drug or treatment may not alter the total hydroxyproline expressed per
mg of muscle, but may nevertheless alter the distribution of collagen within the muscle. In
such cases, point-counting procedures for assessing the proportion of collagen in muscle
cross-sections and morphometric evaluation of the density and area of collagenous fibrotic
plaques provide an independent measure to exclude the possibility that a treatment alters
collagen distribution without altering total collagen expression.

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3 CAUTIONS

Age, Muscle, and Gender Considerations. Since collagen levels increase with age in skeletal
muscle from both nondystrophic and dystrophic mice, it is imperative that comparisons be
made only between groups that are appropriately age-matched. Collagen levels also differ
between different muscles in both nondystrophic and mdx mice. It is therefore quite important
to identify the muscle being evaluated. Experiments comparing hydroxyproline contents in
adult gastrocnemius and costal diaphragm muscle indicate that hydroxyproline content is not
significantly elevated over nondystrophic levels in the gastrocnemius, but is quite
substantially elevated in the costal diaphragm (Graham et al., 200X; Fig. 2). These results and
those of Stedman et al (1991) clearly identify the costal diaphragm as the most appropriate
muscle to use when examining the potential efficacy of experimental treatments to reduce
fibrosis in the mdx mouse. We have not observed obvious gender differences in
hydroxyproline content in either nondystrophic or mdx mice.

Purity of muscle sample. In determining hydroxyproline content, it is essential that only

muscle tissue be used, and that all tendinous material be carefully removed from the sample
before performing the assay. A good sample would be obtained by using approximately ½ of
an entire costal hemidiaphragm. The muscle should be flash-frozen and may be stored at -80O
C for several weeks before running the assay. In practice, samples are usually run within
about 4 weeks of the time that they are isolated.

Critical steps in the hydroxyproline assay. There are some particularly important steps in the
hydroxyproline assay that must be monitored carefully to ensure quality results.
(Steps 2A and B).The first two steps in the hydrolysis (step 2A, B in Methods)
°
of the muscle tissue involve overnight incubation of the sample in 5 N HCl at 130 C. The acid
solution must be contained within stoppered glass tubes that do not leak. Therefore, the
volume of acid in each tube should be visually monitored before and after the incubation
period to ensure it remains constant throughout the incubation period. Instances in which
there is a clear loss of volume indicate a gas leak and should result in termination of the
experiment for that sample. To avoid this problem, use brand new screw-tops and make sure
that the screw tops are tightly applied to each tube prior to the incubation.
(Step 4N). In removing the toluene layer containing the final pyrrole reaction
product, it is imperative not to disturb or remove any of the aqueous layer. If aqueous solution
is removed, it clouds the colorimetric reaction measured in step 5. To avoid this problem,
remove only 1.5 ml of the 2.5 ml toluene layer and make the appropriate correction to
determine the hydroxyproline level in the 2.5 ml of toluene extract (Evaluation and
Interpretation of Results).

Continuity of assay. Theoretically, once the muscle sample is fully hydrolyzed (Step 2c), it
may remain at room temperature for up to 3 to 4 hours before processing the sample (Step 4).
Once the processing steps have begun (Step 4A), they should continue without interruption
until the assay has been completed (Step 5). In practice, it takes about 5 hours to fully process
about 30 test tubes and the process is facilitated by having more than one investigator engaged
in Steps 4 and 5.

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Precautions. Standard protection (gloves, eye protection, lab coat) should be used at all times
when handling acids and toluene solutions. Toluene extractions should be performed under a
fume hood. All solutions at the end of the procedure should be discarded using
environmentally-appropriate procedures for handling acids and organic solvents.

4 MATERIALS

1) Stock Buffer Composition:

50 g citric acid monohydrate (Sigma #C7129)

12 ml glacial acetic acid (Sigma #A6283)
120 g sodium acetate trihydrate (Sigma #S7670)
34 g NaOH
Total volume in ddH2O (dd means double distilled or filtered and distilled) = 1 liter

Measure 900 ml ddH2O and add 12 ml glacial acetic acid slowly while mixing. Add
citric acid monohydrate, sodium acetate trihydrate, and NaOH. Add ddH2O to total
volume of 990 ml and adjust pH to 6.0 with HCl and NaOH as needed. Add ddH2O to
total volume of 1 liter and check pH again. Label (HP Stock Buffer solution, pH,
initials, date) and store in refrigerator.

2) Ehrlich’s Reagent (from Prockop and Udenfriend, 1960; for step 4P):

Slowly mix 27.4 ml of concentrated sulfuric acid to 200 ml of absolute alcohol in a

500 ml beaker.
In another beaker, add 120 g of p-dimethylaminobenzaldehyde to 200 ml of absolute
alcohol.
Slowly mix the two solutions in the first beaker.
Store the final solution in the refrigerator, and allow crystals to come out of the
solution before using.

Other chemicals needed:

Phenolphthalein solution (1%)

0.8 N and 0.1N KOH
0.1 M sodium borate buffer (pH 8.7)
0.2 M chloramine-T
3.6 M sodium thiosulfate
KCl
Toluene

Instruments needed for measurements:

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Glass test tubes (15 ml capacity) with screw caps

Oven
Spectrometer
Tabletop centrifuge
Tabletop test tube Invertor
Tabletop vortexor

5 METHODS

1) Tissue preparation

Blot and weigh fresh or thawed muscle sample (e.g. ½ to 1 costal hemidiaphragm) to
obtain wet weight. Make sure that the muscle sample is carefully trimmed and contains no
tendinous material.

2) Hydrolysis. Always wear appropriate protection (lab coat, heavy rubber gloves, eye
protection) and use standard safety procedures when handling acids.

A. Add 5N HCl (1ml HCl /10mg tissue) to the muscle sample in glass test tube. Tighten
screw-tops carefully and ensure that the screw-tops are not over-worn from previous
use.

B. Incubate tubes at 130oC for 12 hrs (setting is number 13 on the Fisher Scientific
Isotemp oven).

C. Remove muscle samples from oven and cool to room temperature. Do not open the
test tubes until they are cooled to room temperature.

3) Prepare chemicals and solutions

A. Determine how many tubes (n) will be run. This includes all the experimental
samples (in triplicate) and the hydroxyproline standards (in duplicate) plus the blank
tube.

B. Weigh approximately 1.5 g of KCl for each test tube and place in weigh boats (see
step 4G).

C. Prepare chloramine T solution (for step 4E):

1) Determine the total volume of chloramine T solution that will be needed.

This is equal to 2n +2 in ml (extra 2 ml for making sure there is enough volume).

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2) Weigh out enough chloramine T for the total volume that is required. Based
on the molecular weight of chloramine T (Sigma # C9887; 227.6 g/mole):

(2n +2) ml x 0.2 moles/1000 ml x 227.6 g/mole = Amount Chloramine

T needed in g.

Example: for 10 tubes to be run:

(20 +2) ml x 0.2 moles/1000ml x 227.6 g/mole = 1.00 g chloramine T

3) Dissolve chloramine T in the appropriate volume of buffer:

a.) Prepare the following volumes:

Volume of Stock Buffer (see Materials) = 0.5*(2n +2)

Volume of ddH2O = 0.2*(2n+2)
Volume of methylcellosolve (2 methoxyethanol; Sigma
#284467) = 0.3*(2n+2)

b.) Instructions for preparing solution:

Add the appropriate volume of ddH2O to the amount of

chloramine T determined in step C2, and then the appropriate volumes of the
stock buffer and methyl cellosolve. Mix thoroughly.

4) Sample and standard processing. To ensure safety, all of the following steps should be
conducted using a fume hood and standard laboratory safety precautions required for
handling acids and organic solvents.

A. Make sure hydrolyzed muscle sample at room temperature is mixed well (invert
closed tube to mix). Remove 50 µl of the hydrolyzed muscle sample, which represents
0.5 mg of the original muscle wet weight, and add to 2.25 ml of ddH2O (need total
volume of 2.3 ml) in glass test tube. Repeat in triplicate for each muscle sample. In a
test tube marked “0” (blank), add 2.3 ml ddH2O. Screw a cap on each test tube.

B. Set up the hydroxyproline standards in 2.3 ml of ddH2O (run each in duplicate)

containing, for example, 0.75, 1.5, 3.0, and 6.0 µg of hydroxyproline. A 1mg/ml stock
solution of hydroxyproline (in ddH2O) may be stored in the freezer. Screw a cap on
each test tube.

C. Add 1 drop of phenolphthalein solution (1%) to each tube and adjust pH to a “faint
pink color” with 0.1 N KOH (Using a Pasteur pipette, add about 5 drops of 0.8 N
KOH then add drops of 0.1N KOH until faint pink).

D. Add 0.5 ml of 0.1 M sodium borate buffer (pH 8.7) to each tube, screw a cap on each
tube, and mix by vortexing at low speed.

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E. Add 2.0 ml of freshly prepared 0.2 M chloramine-T solution to each tube, screw a cap
on each tube, and incubate at room temperature for 25 minutes.

F. Add 1.2 ml of 3.6 M sodium thiosulfate to each tube, screw a cap on each tube, and
mix thoroughly (vortex) for 10 sec.

G. Add 1.5 g KCl and 2.5 ml toluene to each tube. Screw a cap on each tube.

H. Shake sample for 5 minutes or slowly invert test tubes 100 times, preferably using a
test tube invertor.

I. Centrifuge at approximately 300-400 g (1 minute) to separate layers.

J. Remove toluene phase using clean Pasteur micropipette. Discard using appropriate
procedures for organic solvents.

K. Heat remaining aqueous phase in boiling water for 30 minutes (cap tightly).

L. Cool to room temperature.

M. Add 2.5 ml toluene to each tube, screw a cap on each tube, and repeat steps 4H-I.

N. Remove 1.5 ml of toluene layer and place in a separate test tube. Screw a cap on each
tube.

O. Gently warm stock Ehrlich’s reagent (see Materials) that is kept in refrigerator in order
to remove crystals.

P. Add 0.6 ml Ehrlich’s reagent, mixing well to prevent layering of the solvent. Incubate
at room temperature for 30 minutes.

5) Measure samples

Using a conventional glass or quartz cuvette, measure the blank-corrected (sample – blank)
absorbance (560 nm) for each solution obtained in step 4P.

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6 EVALUATION AND INTERPRETATION OF RESULTS

A. Perform a linear regression analysis of the results using the hydroxyproline standards to
identify the relationship between hydroxyproline (µg) and Absorbance (e.g., Fig. 1).

Figure 1. Example of a
0.4 hydroxyproline standard curve
y = -0.00921 + 0.0572 x and the least squares
Absorbances (560 µm)

R = 0.997 determination of the standard

0.3
curve equation relating
hydroxyproline (µg) to
0.2 Absorbance. Inset shows the
results of regression analyses.
0.1

0.0
0 1 2 3 4 5 6 7

Hydroxyproline (µg)

B. Determine the amount of hydroxyproline (µg) in each sample test tube (Step 4P) using the
regression curve from the hydroxyproline standards. In the example shown (Fig. 1), the
appropriate relationship is:
Sample Hydroxyproline (µg) = (Sample Absorbance + 0.00921)/0.0572

C. Since the amount of hydroxyproline in the final colorimetric reaction (Step 4P) represents a
proportion (1.5 ml/2.5 ml) of the total hydroxyproline in the final toluene extract (Step 4M),
multiply the result obtained in Step 6B by (2.5/1.5) to obtain the total amount of
hydroxyproline present in the final extract.

D. Divide the result obtained in Step 6C by the amount of muscle (wet weight) contained in
the initial sample (0.5 mg; Step 4A) to obtain the hydroxyproline content (µg
hydroxyproline/mg muscle). Examples of typical values obtained from the costal diaphragms
of 14 month old nondystrophic and mdx mice are shown (Fig. 2).

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N=6 Figure 2. Hydroxyproline Contents (µg

Hydroxyproline Content (µg/mg)

25 ** hydroxyproline/mg muscle) determined from

the costal diaphragms of 14 month
20 nondystrophic (gray histobar) and mdx (gray
hatched histobar) mice. N is the number of
15 preparations (mice). Shown are the means
N=5 and standard errors. ** indicates p< 0.01
10 between mean nondystrophic and mdx
values.
5

0
Nondystrophic MDX

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7 REFERENCES
Graham KM, Singh R, Millman G, Malnassy G, Gatti F, Berge J, Bruemmer K, Stefanski C,
Curtis H, Sesti J, and Carlson CG. Inhibitors of the NF-κB pathway do not influence collagen
deposition in dystrophic (mdx) muscle, submitted, 200X

Prockop D.J. and Udenfriend S., (1960). A specific method for the analysis of hydroxyproline
in tissues and urine. Analytical Biochemistry, 1, 228-239.

Stedman, H.H., Sweeney, H.L., Shrager, J.B., Maguire, H.C., Panettieri, R.A., Petrof, B.,
Narusawa, M., Leferovich, J.M., Sladky, J.T., Kelly, A.M., (1991). The mdx mouse
diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature
352, 536-539.

Switzer, B.R., Summer, G.K., (1971). Improved method for hydroxyproline analysis in tissue
hydrolyzates. Anal Biochem. 39(2), 487-491.

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